Pescadillo, which has been found to be involved in the process of ribosomal biogenesis, has been demonstrated to play a role in embryonic development, DNA replication, and gene transcription. While deregulation of ribosomal biogenesis was also found to contribute to carcinogenesis, and proteins that regulate ribosomal biogenesis are commonly overexpressed in primary tumors, little is known about the clinical significance and biological function of pescadillo in human breast cancer. In the current study, we found that the expression of pescadillo was markedly up‐regulated in human breast cancer cells and tissues at both mRNA and protein levels. Immunohistochemical analysis revealed that pescadillo expression in clinical stage I–IV primary breast cancer tissues was statistically significantly higher than that in normal breast tissues (P < 0.05). Furthermore, we demonstrated that knockdown pescadillo with RNAis inhibited cell proliferation and the colony‐forming ability of the cells. Anchorage‐independent growth ability assay indicated that ablation of pescadillo led to the reduction of breast cancer cells tumorigenicity in vitro. Moreover, depletion of endogenous pescadillo resulted in decreased expression of cell cycle protein cyclin D1 and up‐regulation of cyclin‐dependent kinase inhibitor p27^Kip1, as well as attenuated protein kinase B (Akt)/glycogen synthase kinase 3 beta (GSK‐3β) signaling. Taken together, our results suggest that pescadillo might play a role in promoting the proliferation and carcinogenesis of human breast cancer, and thereby might be a potential target for human breast cancer treatment. (Cancer Sci 2009; 100: 2255–2260)